Summary of Work. Background. We aim to elucidate the molecular mechanisms that control the assembly of viruses with the ultimate goals of defining targets for antiviral compounds, characterizing viral antigenicity, and understanding the assembly of macromolecular complexes in general. This research focuses on the large-scale conformational changes that accompany capsid maturation and on the interaction of virions with host cells. Our major progress over the past year is as follows: (1) Hepatitis B Virus Capsid (Core Antigen). Despite the availability of effective vaccines, HBV remains a public health problem of immense proportions, motivating further efforts to elucidate its replicative cycle. We have been studying its capsid protein (core antigen), which we first found to form dimers that are capable of self-assembly into particles of two different sizes - one with T=3 icosahedral symmetry (90 dimers; 28nm diameter), the other with T=4 (120 dimers; 34nm diameter). We also found that the capsid protein of HBV, unlike most other viruses, is predominantly alpha-helical. In 1997, we calculated a density map from cryo-electron micrographs at the unprecedentedly high resolution of 0.9nm, in which much of the secondary structure was directly visible, including the 4-helix bundle that forms the dimerization motif. Our subsequent research (FY98) used high resolution labelling techniques to establish the precise locations of both termini and an internal hexapeptide (res. 78-83), thus delineating the overall path of the polypeptide chain through our density map. The implied fold ? a novel one for a capsid protein - has been confirmed by X-ray crystallography from another laboratory in the past few months. In FY99, our main effort on HBV has been to further refine the sensitivity of our heavy metal cluster labelling techniques in order to be able to use them more widely and more incisively in studies of other complexes. Our first success was with an 11-atom gold cluster, undecagold, attached to the C-terminus of core antigen. More recently, we have been able to visualize an even smaller label, the 4-atom tetrairidium cluster, at the same site. (2) Capsid Assembly and Maturation of Herpesviruses. Herpesviruses constitute a widely diversified family of large, complex, DNA viruses. Eight members of the family cause diseases in humans, including skin diseases. We have studied the elaborate process of herpesvirus capsid assembly for several family members, including herpes simplex type 1, simian cytomegalovirus, and ? in the last year - Kaposi sarcoma-associated herpesvirus. In previous work, we combined electron microscopy with biochemistry and genetics to define the molecular anatomy of herpesviruses and to characterize the individual roles of the six major capsid proteins in assembly. These properties have turned out to be closely conserved among different herpesviruses, despite wide evolutionary divergence at the sequence level, and quite distinct from those of any other known family of viruses. Nevertheless ? to our surprise - we have identified a growing number of qualitative similarities between the respective assembly pathways of herpesviruses and DNA bacteriophages. In FY99, our main focus has been to further characterize the HSV-1 procapsid, a precursor particle that differs markedly in structure, composition, and stability from the mature capsid. In FY97, we discovered this particle in in vitro assembly experiments performed using crude homogenates of insect cells infected with recombinant baculoviruses expressing herpesvirus capsid proteins. In FY99, we have established the morphogenic competence of these proteins by assembling identical particles in vitro from purified proteins, and confirmed their status as obligatory precursors and by isolating such particles directly from infected cells. The latter experiments were performed with mutants in the viral protease for which, procapsids accumulate in infected cells: in contrast, procapsids are rare in wild-type infections because they mature shortly after they are assembled. Our focus is now turning to dynamic aspects of procapsid maturation. (3) Structure, Assembly, and Maturation of Bacteriophage Capsids. Our primary interest in bacteriophage capsid assembly lies in the monumental conformational changes that accompany maturation of precursor capsids. These changes are irreversible, frequently involve partial refolding of the subunits, and are stringently controlled. As such, they afford unique opportunities for insight into large-scale conformational changes in proteins. Although phages vary widely in size, sequence, and other properties, the maturation transformation is a universal feature and we study this behavioral event in several systems to exploit expedient aspects of each. In FY99, studies on T4 initiated over the preceding three years have come to fruition. T4 is distinguished by its large size, order of complexity, and in the binding of two accessory proteins to its outer surface. One of these proteins, gpSOC (12 kDa), stabilizes the capsid by binding in a clamp-like fashion over intermolecular interfaces. We have now been able to visualize the binding of both of these proteins to the T4 capsid surface. The corresponding protein of bacteriophage lambda is called gpD. Motivated by the very recent crystal structure determination of gpD by colleagues at FCRF-Frederick, we isolated lambda capsids and characterized their structure at 1.5 nm resolution by cryo-microscopy. Our map clearly shows gpD trimers bound over the trigonal sites, establishing that the crystal structure represents the biologically active state of the protein and revealing its mode of binding. Further productive exploitation of crystallographic data is central to our most recent work on the HK97 system, whose mature capsid has been solved by colleagues at Scripps Institute (and which, in this instance, has no accessory proteins: rather this capsid is stabilized by covalent crosslinks). We have determined the precursor capsid structures to ~ 1.2 nm resolution by cryo-microscopy and are combining the two sources of information with the aim of developing an account of the maturation-related conformational changes in atomic detail.(4) Interaction of Poliovirus with Host Cell: Receptor Binding. When a picornavirus such as the human pathogen, poliovirus, encounters a susceptible cell, the virus binds to a cellular receptor. Unlike enveloped viruses, which enter cells by fusing with the cell membrane or endocytosis, poliovirus entry appears to be mediated by changes in its capsid that follow its interaction with the receptor and lead to discharge of its RNA into the host cell. In the culmination of a project pursued over the preceding five years, we have completed our structural analysis of two conformers of poliovirus that are implicated in the transfer process and have submitted for publication a paper describing these results and their implications. Again, this study involved a combination of crystallography and cryo-microscopy, whereby the crystal structure of poliovirus (known since 1985) was systematically adjusted to fit our cryo-microscopic models of the two cell-entry states of the virus. The resulting changes appear to emulate, in miniature, the movements of tectonic plates in an earthquake as the rigid beta-barrel domains of the consid proteins shift, creating gaps through which the RNA may be released. We have also received consignments of a soluble form of the receptor ? a 3-domain IgG-like molcule, and studied in detail its mode of binding to the virus particle.(5) Amyloid-like Deposits of HIV gp41 Ectodomain Trimers Implicated in AIDS-Related Dementia. The condition of demented AIDS patients has been correlated with the deposition in neurological tissue of insoluble aggregates of the gp41 ectodomain of the HIV envelope glycoprotein. The structure of the gp41 trimer is known: each subunit consists of two alpha-helices connected by a loop. One set of three helices forms a coiled-coil and the other three, oppositely oriented, helices pack round them. In this new project, pursued within the aegis of the IATAP program, we have studied these aggregates by electron microscopy, using a combination of conventional negative staining, mass determinations by dark-field scanning transmission electron microscopy (STEM) of unstained specimens, and unconventional negative staining, whereby vanadate-embedded material is visualized by dark-field STEM. Taken together, these observations imply that aggregates consist of variable numbers (approximately 7 to 70) of gp41 trimers, associated by means of interactions among their connecting loops which associate at the core (interior) of the aggregate. The packing arrangement is not very regular but the trimers are predominantly oriented with their long axes oriented radially. The pathogenesis of these aggregates may relate to that of amyloid ? indigestible aggregates of proteins that accumulate in a number of disease states, with the distinction that although unlike conventional amyloid, which is rich in beta sheets, the gp41 aggregates are primarily alpha-helical.